Method of producing salinomycin antibiotics

ABSTRACT

Polyether type antibiotics are produced by culturing a polyether type antibiotic-producing microorganism is a medium containing a fatty acid or its precursor and ammonia or an ammonium salt or urea.

BACKGROUND OF THE INVENTION

The present invention relates to an improvement in a method of producingpolyether type antibiotics in an industrial scale.

As polyether type antibiotic there have been known Monensin (Journal ofAmerican Chemical Society, Vo. 89, page 5757, 1967), X-206 (ChemicalCommunications, 927, 1971), Salinomycin (British Pat. No. 1,378,414),SY-1 substance (Japanese O.P.I. No. 86191/76), SY-2 substance (JapanesePatent Application No. 5762/77), 4-methylsalinomycin (A 28086 substance)(Japanese O.P.I. No. 9788/76), Lasalocid (Journal of American ChemicalSociety, Vol. 73, 5295, 1951), Dianemycin (Journal of Antibiotics, Vol.22, page 161, 1969), Nigericin (Biochemical and Biophysical ResearchCommunication, Vol. 33, page 29, 1968), A-204 A (Journal of AmericanChemical Society, Vol. 95, 3399, 1973) and the like, and among these,Salinomycin, 4-methylsalinomycin, SY-1, SY-2, SY-3, SY-4, SY-5, SY-6,SY-7 and SY-8 substance are called Salinomycin type antibiotics becausethey have the similar chemical structures.

In this invention, the term "salinomycins" means each compound, or anymixture of at least two compounds, selected from the group consisting ofsalinomycin, SY-1, SY-2, SY-3, SY-4, SY-5, SY-6 and the like.

The present inventors already found that salinomycin, SY-1 and SY-2 wereproduced in the culture of Streptomyces albus waxman and henrich No.80614 strain (FERM-P. No. 419), and succeeded in isolating theantibiotics from the culture (British Pat. No. 1378414, JapaneseUnexamined Patent Publication No. 86191/1976 and Japanese PatentApplication No. 5762/1977).

The inventors continued the study and found that, when the above strainis cultured in the medium containing fatty acid or its precursor, itproduces salinomycin, SY-1, and SY-2 in high yield, and also producesnew compounds such as SY-3, SY-4, SY-5, SY-6, SY-7 and SY-8.

As described in said literatures, these are produced by culturing eachantibiotic producing microorganism belonging to the genus ofStreptomyces. However, the yield of each antibiotic produced by suchknown method is not always satisfactory.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method ofproducing polyether type antibiotics in remarkably high yields withindustrial advantages.

Another object of the present invention is to provide a method ofproducing Salinomycin type antibiotics such as salinomycin,4-methylsalinomycin, SY-1, SY-2, SY-3, SY-4, SY-5, SY-6, SY-7 and SY-8substances in high yield.

The foregoing and other objects of the present invention have beenattained by culturing a polyether type antibiotic-producingmicroorganism in a medium containing a fatty acid or its precursor andammonia or an ammonium salt or urea.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the special feature of the present invention, salinomycin typeantibiotics such as salinomycin, 4-methylsalinomycin, SY-1, SY-2, SY-3,SY-4, SY-5, SY-6, SY-7 and SY-8 substances are produced by culturing asalinomycin type antibiotic-producing microorganism in a mediumcontaining a fatty acid or its precursor and ammonia or an ammonium saltor urea.

The fatty acids used in the present invention are saturated orunsaturated fatty acids, for example, acetic acid, propionic acid,caproic acid, capric acid, palmitic acid, stearic acid, methacrylicacid, undecylic acid, and particularly linolic acid, linolenic acid oroleic acid being preferable. The precursor of fatty acid means asubstance that is capable of giving said fatty acid outside or insidethe microorganism cell, such as mono-di- or triglycerides of fatty acid,esters of fatty acid or salts of fatty acid. Further, there can be usedsoy bean oil, safflower oil, cotton seed oil, sesame oil, olive oil,rape oil, peanut oil, maize oil (corn oil), sunflower oil and likevegetable oils, code oil and like fish oils and lard and like animalfat-and-oils, which contain said precursors.

The esters of fatty acid can be C₁ -C₁₈ alcohols of said fatty acid.

The salts of fatty acid can be ammonium salt and alkali metal salts andalkaline earth metal salts of said fatty acid.

The addition amount is generally about 1-25%, particularly about 12-20%based on the medium.

Ammonia is used in gaseous form or in the form of aqueous solution. Asammonium salt there are used ammonium salts of inorganic acids such ashydrochloric acid, sulfuric acid, nitric acid and phosphoric acid ororganic acids such as acetic acid, propionic acid, higher fatty acid,oxalic acid, tartaric acid, hydrogentartaric acid, citric acid, lacticacid and malic acid. The addition amount is generally about 0.1-1.0%particularly about 0.3-0.5% based on the medium.

As for the time for addition of these additives, addition is effectiveso long as production of potency of polyether type antibiotic continues,and is conducted at any time either before or after beginning ofcultivation. Although the cultivation conditions of the presentinvention can be selected in accordance with the methods described insaid known literatures, excepting that fatty acid or its precursor isused as major carbon source and that ammonia or ammonium salt is used asessential component of the medium, it is possible to raise theproduction efficiency by varying the conditions depending on the kind ofantibiotics.

The microorganism used in the present invention include generallypolyether type antibiotics producing strains belonging to the genus ofStreptomyces as well as the strains described in said literatures andtheir natural or artificial mutants.

Separation and purification of the products can be carried out inaccordance with known methods. Since the objective substance is oftencontained mainly in the solid portion containing the cells when theproduction amount of the objective substance is abundant, it isdesirable to vary the extraction step properly in order to heighten therecovery percentage of the objective-substance from the solid portion.It is possible to use the objective compound in the state that it iscontained in the solid portion depending on the use purposes, withoutseparating the objective substance from the solid portion.

According to the present invention the production amount of polyethertype antibiotics, particularly Salinomycin type antibiotics can beremarkably increased. For example, the yield of Salinomycin is generally100-300 γ/ml in known method, whereas the yield is about 10,000-20,000γ/ml in the medium containing fatty acid or its precursor, and the yieldis further increased to about 50,000-80,000 γ/ml when said medium isfurther added by ammonia or ammonium salt.

The preferable feature of the present invention to produce salinomycintype antibiotics will be further illustrated.

According to the method of this invention, salinomycins mainly occur inthe mycerial mass, at it is preferable to recover salinomycins from themycelial mass. In addition to salinomycin, SY-1, and SY-2, another newcompounds, especially SY-3, SY-4, SY-5, SY-6, and the like are obtainedfrom the culture.

The strains used in this invention include Streptomyces albus No. 80614and its mutants artificially or naturally produced, as well as the otherStreptomyces strains capable of producing salinomycins. However, some ofthe salinomycins can occasionally not be detected in the

    __________________________________________________________________________    Characteristics of salinomycins                                               Characteristics                                                                       Rf values***                                                                  Solvent system                m.sup.+.m/e                                                                        antimicrobial                              CHCl.sub.3 2O                                                                       EtOAc 4           Vanillin**                                                                          (methyl                                                                            activity***                        Factor  MeOH 1                                                                              (CH.sub.3).sub.2 CO 1                                                                EtOAc                                                                             10% H.sub.2 SO*.sub.4                                                                reagent                                                                             ester)                                                                             100 γ/ml                                                                       Remarks                     __________________________________________________________________________    SY-1    0.50  0.95   0.83                                                                              Yellow       748  21                                 Salinomycin                                                                           0.40  0.90   0.60       Red   764  30                                 SY-2    0.35  0.35   0.15                                                                              Yellow 748    20                                     SY-3    0.45  0.92   0.78                                                                              Yellow       748  28     (SY-1 analogue)             SY-4    0.26  0.30   0.13       Red   764  25     (Salinomycin analogue)      SY-5    0.17  0.19   0.10       Red   766  20     (18, 19-dihydrosalino-                                                        mycin analogue)             SY-6    0.05  0.06   0.03       Red   782  15     (18, 19-dihydrosalino-                                                        mycin analogue)             SY-7    0.80  0.98   0.95                                                                              Yellow            17     (SY-1 analogue)             SY-8    0.11  0.13   0.06                                                                              Yellow            14     (SY-1 analogue)             SY-1:   20-deoxy-salinomycin                                                  SY-2:   stereoisomer of SY-1                                                  SY-4:   5-hydroxy-salinomycin                                                 SY-5:   18, 19-dihydro-salinomycin                                            SY-8:   stereoisomer of SY-5                                                  __________________________________________________________________________     *Spray on silica gel plate (room temperature)                                 **Dissolve 3 g of vanillin in 100 ml methanol. Add 0.5 ml of H.sub.2          SO.sub.4 with stirring. Spray on silica gel plate. Heat for 5 min at          60° C.                                                                 ***Silica gel TLC (Wakogel B10, thickness 0.25 mm)                            ****diameter of inhibitory zone test organism: Batilus subtilis          

culture, depending on the strain and fermentation conditions.

Fermentation conditions employed in this invention can be any onecommonly used for culturing Actinomycetes, except that main carbonsource should be a fatty acid or its precursor. Maximum production ofsalinomycins usually occurs after 150 to 260 hours from the start offermentation. Ratio of each of salinomycins produced sometimes variesdepending on the incubation time. Naturally, the composition of mediumand fermentation conditions should be decided for each strain andexternal conditions, so that most desirable results are obtained.

Salinomycins can be isolated from the culture medium by utilizing thephysico-chemical properties of salinomycins. Because salinomycins arestructurally related to each other, the known extraction methods forsalinomycin and SY-1 can be applied to the isolation of salinomycins,however, as for salinomycin, because a large portion of salinomycin iscontained in mycelial mass, the extraction process is preferablymodified so as to increase the recovery rate of salinomycin. Forexample, it is preferred to adjust the whole fermentation broth to pH2.0-6.0 to precipitate salinomycin, and then to extract the mycerialmass together with the precipitate thus formed with an organic solvent.Among the preferred solvents are acetone, ethyl acetate, butyl acetate,n-hexane, chloroform and the like. After applying the solution to theabsorptive materials suitable for the absorption of salinomycins, thesalinomycins are eluted by a suitable solvent system.

the effluent is collected in 2-5 fractions according to the purpose.Separation and purification of salinomycins are effected by theprocedures utilizing the difference between the properties of desiredcompound and impurities. Namely, chromatography, solvent extraction andthe like are repeated for each fraction to give salinomycin, SY-1, SY-2,SY-3, SY-4, SY-5, SY-6, SY-7, SY-8, and the like, individually or asmixtures. The resulting products can be further purified byrecrystallization or chromatography.

The properties of salinomycins obtained by the process of this inventionare shown in the following table.

According to the preferred feature of this invention salinomycin, SY-1,and SY-2, especially salinomycin are obtained in surprisingly higheryield than the known process. New compounds, SY-3 through -8 are alsoobtained according to this invention. They are comparable to salinomycinin their activities against microorganisms and useful as medicines.Also, because of their structural similarity to salinomycin, theirusefulness as veterinary medicines looks very likely.

REFERENCE EXAMPLE 1

Streptomyces albus waxman and henrich No. 80614 strain (FERM-P. No. 419)is inoculated to a medium containing glycerine 2.0%, peptone 0.5% andmeat extract 0.5% and cultured at 33° C. for 48 hours under shaking. 1liter of this culture liquid is inoculated to 100 liters of liquidmedium (200 liter tank made of stainless steel) containing glucose 2%,starch 1%, soy bean powder 2.5%, beer yeast 0.4%, meat extract 0.1%,sodium chloride 0.2% and antifoaming agent KM-68-2F (product of ShinetsuChemical Industry Co., Ltd., silicone type) 0.1% and cultured at 33° C.for 144 hours under stirring with the aeration volume of 100 l/m. Thereis obtained a culture liquid containing 100-300 γ/ml of Salinomycin.

REFERENCE EXAMPLE 2

The 80614 strain which is the same strain as described in Example 1 isinoculated to a medium containing glycerine 2.0%, peptone 0.25%, meatextract 0.5% and edible salt 0.1%, and cultured at 33° C. for 48 hoursunder shaking.

This culture liquid is an amount corresponding to 1% of the medium isinoculated to the medium containing glucose 4.0%, soy bean powder 1.0%,beer yeast 1.0% and calcium carbonate 0.2%, and cultured for 30 hours at33° C. under shaking, and this culture liquid is made the second-stagepre-culture liquid. One liter of this second-state preculture liquid isinoculated to 100 liters of liquid medium containing soy bean oil 10%,glucose 1.0%, soy bean powder 1.0%, calcium carbonate 0.5%, potassiumsecondary phosphate 0.01%, and cultured for 210 hours at 33° C. with anaeration volume of 100 l/m under stirring. There is obtained a cultureliquid containing 20,000 γ/ml of Salinomycin.

EXAMPLE 1

(A) Production of salinomycin

The 80614 strain which is the same strain as described in Example 1 isinoculated to a medium containing glycerine 2.0%, peptone 0.25%, meatextract 0.5% and edible salt 0.1%, and cultured at 33° C. for 48 hoursunder shaking. This culture liquid in an amount corresponding to 1% ofthe medium is inoculated to the medium containing glucose 4.0%, soy beanpowder 1.0%, beer yeast 1.0% and calcium carbonate 0.2%, and culturedfor 30 hours at 33° C. under shaking, and this culture liquid is madethe second-stage, pre-culture liquid. One liter of this second-stagepre-culture liquid is inoculated to 100 liters of liquid mediumcontaining soy bean oil 10%, glucose 1.0%, soy bean powder 1.0%, calciumcarbonate 0.5%, potassium secondary phosphate 0.01%, and cultured for210 hours at 33° C. with an aeration volume of 100 l/m under stirring.There is obtained a culture liquid containing 20,000 γ/ml of salinomycin(salinomycin only).

(B) Separation and purification of salinomycin, SY-1 and SY-2.

The culture liquid obtained in (A) is adjusted to pH 4.5-5.0 with dilutehydrochloride, admixed with 4% by weight per volume of filter aid withstirring, and filtered. The filtrate (80 liters) is extracted with 50liters of butyl acetate with stirring. Mycelial mass is extracted with30 liters of butyl acetate. Both butyl acetate solutions are combinedand washed with 20 liters of 5% aqueous sodium bicarbonate solution. Oneliter of the washed butyl acetate solution is concentrated under vacuumto dryness to give 250 g of crude powder of salinomycin containing traceamount of SY-1 and SY-2.

Fifty grams of the crude salinomycin powder is dissolved in ethylacetate and applied to column (50 g of active almina, commercial productof Wako Junyaku Co.). After washing the column with one liter of ethylacetate, salinomycin and SY-1 are eluted with a 100:5 mixture of ethylacetate-methanol solution. The fractions containing salinomycin and SY-1are combined and concentrated. The concentrate is diluted in 50 ml ofchloroform-methand solution (100:2) and applied to the column of 300 gsilica gel (Wakogel-200, Wako Junyaku Co.) packed in the same solventmixture. Elution is carried out with the same solvent mixture to givepure salinomycin fraction and pure SY-1 fraction. Each fraction isconcentrated and crystallized from acetone-water solution to give 5 g ofsalinomycin and 30 mg of SY-1 in pure crystals, respectively.

The almina column as mentioned above is further developed with a 100:15mixture of ethyl acetate-methanol to elute SY-2, which is separated andpurified by silica gel chromatography in the same manner as describedabove, and crystallized from acetone-water solution to give 3 mg of purecrystalline SY-2.

EXAMPLE 2

(A) Salinomycin is cultured in the same manner as described in Example1.

(B) Recovery of salinomycins

The culture obtained in (A) is adjusted to pH 4.5-5.0, heated at 60° C.for 10 minutes with stirring, admixed with 4% by weight per volume offilter aid with stirring and filtered. The resulting mycelial masscontains salinomycins and SY group compounds in high yield.

(C) Separation of salinomycins complex

Mycelial mass obtained in (B) is extracted twice with 50 liters of ethylacetate. The extracts are combined and washed with 50 liters of 5%aqueous sodium bicarbonate solution. Ethyl acetate phase is concentratedunder vacuum to dryness to yield 7 kg of crude salinomycins complexcontaining SY group compounds.

(D) Separation and purification salinomycin

Seven kg of crude powder obtained in (C) is dissolved in 100 liters ofhexane, concentrated under vacuum to 20 liters and left at 5° C. to give2.7 kg of crude crystallin salinomycin. After repeating this procedure,recrystallization of salinomycin from hexane gives 2.4 kg of puresalinomycin sodium salt.

(E) Separation and purification of SY group compounds

The mother liquor separated from the precipitate in the process in (A)is concentrated to dryness to give 4.6 kg of complex containing a smallamount of salinomycin and SY group compounds. The complex is dissolvedin 50 liters of hexane-ethyl acetate solvent mixture (2:1) and appliedto the column of 12 kg of almina packed in the same solvent system,developed with 20 liters of a 100:2 mixture of ethyl acetate-methanol toelute SY-3, SY-7 and salinomycin. Two hundred g of the mixture ofsalinomycin and SY-1 is separated by precipitation in the same manner asdescribed in (D). The filtrate containing SY-3 and SY-7 is concentratedto dryness to give 100 g of crude powder.

One hundred g of crude powder thus obtained is dissolved in 300 ml of a100:2 mixture of chloroform-methanol and applied to the column of 6 kgof silica gel (Wakogel C-200, Wako Junyaku Co.), and developed with thesame solvent mixture. Each of SY-3 and SY-7 fractions is concentratedand applied to silica gel thin layer chromatogram (Wakogel B-10 withthickness of 0.25 mm, solvent system; a 20:1 mixture ofchloroform-methanol). SY-3 and SY-7 are collected by scraping off theareas with Rf values 0.45 and 0.80, respectively. The silica gel holdingeach compound is eluted with a 20:1 mixture of chloroform-methanol andthe solvent mixture is removed by evaporation to give 18 mg of SY-3 and35 mg of SY-7 both in pure powders.

The above almina column is eluted with 20 liters of a 5:1 mixture ofethyl acetate-methanol, and the effluent is concentrated under vacuum togive 200 g of crude powder, which contains the complex of SY-2, SY-4,SY-5, SY-6 and SY-8. These compounds are isolated individually by silicagel column chromatography. Two hundred g of the crude powder isdissolved in 500 ml of a 100:2 mixture of chloroform-methanol, appliedto the column of 2 kg silica gel (Wakogel C-200) packed in the samesolvent mixture, and developed with the same solvent mixture to giveeach fraction containing SY-2, SY-4, SY-5, SY-6 and SY-8, respectively.Each fraction is concentrated to dryness to give 20 g of SY-2, 300 mg ofSY-4, 250 mg of SY-5, 800 mg of SY-6 and 100 mg of SY-8 as crudepowders, respectively. Crude SY-2 powders are crystallized from acetonewater to give 15 g of pure crystalline SY-2.

Other crude powders are purified by thin layer chromatography of silicagel in the same manner as employed in the separation and purification ofSY-3 and SY-7 to give 26 mg of SY-4, 20 mg of SY-5, 80 mg of SY-6 and 30mg of SY-8 each in pure powders.

EXAMPLE 3

(A) The second-stage pre-culture liquid of Example 1 in an amountcorresponding 10% of the medium is inoculated to the medium containingglucose 4%, soy bean powder 3%, defatted wheat germs 3.0%, calciumcarbonate 0.2% and antifoaming agent KM-68-2F 0.1%, and cultured for 24hours at 33° C. to give the third-stage pre-culture liquid.

Ten liters of the third-stage pre-culture liquid is inoculated to themedium containing soy bean oil 16%, soy bean powder 0.5%, defatted wheatgerms 1.0%, sodium chloride 0.2%, potassium chloride 0.2%, ammoniumsulfate 0.3%, calcium secondary phosphate 0.02%, magnesium sulfate 0.01%and antifoaming agent KM-68-2F 0.1%, and cultured for 290 hours at 33°C. with an aeration volume of 100 l/m under stirring. The productionamount of salinomycin at the end of cultivation is 60,000 γ/ml(salinomycin only). In this case the similar production amount isattained even when soy bean oil is added in a small amount at thebeginning and then the addition amount is increased. No difference inproduction amount is observed between cases in which silicone type andpolyether type antifoaming agents are used.

(B) Treat the fermentation broth obtained in (A) according to theprocedure of Example 2 (B) to give mycelial mass containing salinomycinsin high yield.

(C) Treat the mycelial mass obtained in (B) according to the procedureof Example 2 (C) to give 16 kg of the complex of SY-1, 2, 3, 4, 5, 6, 7and 8.

(D) Dissolve 16 kg of crude powder obtained in (C) in 100 liters ofhexane and concentrate under vacuum to 40 liters. Then treat thesolution according to the same procedure described in Example 2 (D) togive 7.1 kg of crude crystallin salinomycin and then 6.3 kg of puresalinomycin sodium salt.

(E) The mother liquor separated from crystals in the process in (D) isconcentrated to dryness to give 9.7 kg of the complex containing SY-1through -8. The complex is dissolved in 100 liters of a 2:1 mixture ofhexane-ethyl acetate, applied to the column of 20 kg of almina in thesame manner as described in Example 2 (E) and developed with 30 litersof a 100:2 mixture of ethyl acetate-methanol to elute SY-1, SY-3 andSY-7. The effluent is treated in the same manner as described in Example2 (E) to give 400 g of salinomycin-SY-1 mixture and 130 g of crudepowder containing SY-3 and SY-7.

One hundred and thirty g of said crude powder is dissolved in 400 ml ofchloroform-methanol solution (100:2), treated by silica gel columnchromatography (8 kg of silica gel is used) to give SY-3-andSY-7-fractions. Each fraction is chromatographed over silica gel thinlayer according to Example 2 (E), and after removing the solvent byevaporation, 30 mg of SY-3 and 50 mg of SY-7 are obtained as purepowders.

The above almina column is eluted with 40 liters of a 5:1 mixture ofethyl acetate-methanol and the effluent is concentrated to dryness togive 250 g of crude powder containing the complex of SY-2, SY-4, SY-5,SY-6 and SY-7. The complex is dissolved in 600 ml of chloroform-methanolsolution (100:2), and then treated by chromatography (20 kg silica gel)in the same manner as described above to give 40 g of SY-2, 600 mg eachof SY-4 and SY-5, 1900 mg of SY-6 and 300 mg of SY-8 as crude powders,purification of which according to the procedure of Example 2 (E) gives30 g of crystallin SY-3, 180 mg of SY-4 powder, 120 mg of SY-5 powder,350 mg of SY-6 powder and 90 mg of SY-8 powder, all in pure form.

EXAMPLE 4

The third-stage pre-culture liquid in Example 3 in an amountcorresponding to 10% of the medium is inoculated to the medium (50 ml)containing each fat-and-oil shown in the following Table 12%, soy beanpowder 0.5%, defatted wheat germs 1.0%, sodium chloride 0.2%, potassiumchloride 0.2%, calcium carbonate 0.5%, ammonium solfate 0.3%, potassiumsecondary phosphate 0.02% and magnesium sulfate 0.01%, and cultured at33° C. for 216 hours under shaking. The production amounts ofsalinomycin at the end of cultivation are shown in the following Table.

    ______________________________________                                                        Yield of                                                      Fat-and-oil     salinomycin (γ/ml)                                      ______________________________________                                        soy bean oil    38000                                                         purified soy bean oil                                                                         36000                                                         (Shirashime oil)                                                              Sesame oil      36000                                                         rape oil        35000                                                         safflower oil   39000                                                         olive oil       37000                                                         cod oil         37000                                                         methyl oleate   34000                                                         methyl myristate                                                                              38000                                                         methyl linolate 38000                                                         ______________________________________                                    

From the resulting culture, salinomycins are separated individually andpurified according to the procedures of Examples 1-3.

What is claimed is:
 1. A method of producing salinomycins, whichcomprises culturing a salinomycins-producing Streptomyces microorganismin a medium containing .Iadd.12-25% .Iaddend.fatty acid or its precursorand ammonia or an ammonium salt and recovering the salinomycins from theculture.
 2. The method of claim 1 wherein salinomycin is recoveredtogether with the mycelial mass from the culture.
 3. The method of claim1 or 2 wherein SY-1, SY-2, SY-3, SY-4, SY-5, and/or SY-6 is recoveredfrom the culture.
 4. The method of claim 1, wherein salinomycin,4-methylsalinomycin, SY-1, SY-2, SY-3, SY-4, SY-5, SY-6, SY-7 and/orSY-8 is recovered from the culture.